1. Field of the Invention
The present invention pertains generally to a receptor protein tyrosine kinase (rPTK) ligand. More particularly, the invention relates to a novel ligand, designated VEGF-related protein (VRP) or VH1, which binds to, and stimulates the phosphorylation of, the Flt4 tyrosine kinase receptor (also known as the Sal-S1 receptor) and the isolation and recombinant production of the same.
2. Description of Related Art
The formation of new blood vessels either from differentiating endothelial cells during embryonic development (vasculogenesis) or from pre-existing vessels during adult life (angiogenesis) is an essential feature of organ development, reproduction, and wound healing in higher organisms. Folkman and Shing, J. Biol. Chem., 267: 10931-10934 (1992); Reynolds et al., FASEB J., 6: 886-892 (1992); Risau et al., Development, 102: 471-478 (1988). Angiogenesis is also necessary for certain pathological processes including tumorigenesis (Folkman, Nature Medicine, 1: 27-31 [1995]) and retinopathy. Miller et al., Am. J. Pathol., 145: 574-584 (1994).
While several growth factors can stimulate angiogenesis (Klagsbrun and D""Amore, Ann. Rev. Physiol., 53: 217-239 [1991]; Folkman and Klagsbrun, Science, 235: 442-447 [1987]), vascular endothelial growth factor (VEGF) (Ferrara et al., Endo. Rev., 13: 18-32 [1992]) is a potent angiogenic factor that acts via the endothelial cell-specific receptor tyrosine kinases fms-like tyrosine kinase (Flt1) (Shibuya et al., Oncogene, 5: 519-524 [1990]; deVries et al., Science, 255: 989-991 [1992]) and fetal liver kinase (Flk1) (also designated KDR). Quinn et al., Proc. Natl. Acad. Sci. USA, 90: 7533-7537 (1993); Millauer et al., Cell, 72: 835-846 (1993); Matthews et al., Proc. Natl. Acad. Sci. USA, 88: 9026-9030 (1991); Terman et al., Biochem. Biophys. Res. Commun., 187: 1579-1586 (1992); Terman et al., Oncogene, 6: 1677-1683 (1991); Oelrichs et al., Oncogene, 8: 11-18 (1993). These two VEGF receptors and a third orphan receptor, Flt4 (Pajusola et al., Cancer Res., 52: 5738-5743 [1992]; Galland et al., Oncogene, 8: 1233-1240 [1993]; Finnerty et al., Oncogene, 8: 2293-2298 [1993]) constitute a subfamily of class III receptor tyrosine kinases that contain seven extracellular immunoglobulin-like domains and a split intracellular tyrosine kinase domain. Mustonen and Alitalo, J. Cell. Biol., 129: 895-898 (1995). See also WO 94/10202 published May 11, 1994 and PCT/US93/00586 filed Jan. 22, 1993 (Avraham et al.). These three receptors have 31-36% amino acid identity in their extracellular ligand-binding domains.
Mice deficient in Flt1 (Fong et al., Nature, 376: 66-70 [1995]) or Flk1 (Shalaby et al., Nature, 376: 62-66 [1995]) (generated by gene targeting in embryonic stem cells) have severe defects in vasculogenesis and die in utero at embryonic day 8-9. The phenotype of the receptor-deficient mice differs considerably, however. Mice lacking Flt1 have a disorganized vascular endothelium that extends to the major vessels as well as to the microvasculature, while endothelial cell differentiation appears to be normal. Fong et al., supra. Mice lacking Flk1 have a major defect in the development of mature endothelial cells as well as a severe reduction in hematopoietic cell progenitors. Shalaby et al., supra. Thus, VEGF may act on endothelial cells at more than one stage of vasculogenesis.
Flt4 is also specifically expressed in endothelial cells; it is first observed in day 8.5 mouse embryos in endothelial cell precursors. Kaipainen et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995), Kaipainen et al., J. Exp. Med., 178: 2077-2088 (1993). See also Hatva et al., Am J. Pathol., 146: 368-378 (1995). As development proceeds, Flt4 expression becomes confined to the venous and lymphatic endothelium and is finally restricted to the lymphatic vessels. Consistent with this finding, adult human tissues show Flt4 expression in lymphatic endothelia while there is a lack of expression in arteries, veins, and capillaries. Kaipainen et al., Proc. Natl. Acad. Sci. USA, supra. Clones encoding human and mouse Flt4 have been isolated either by PCR with primers from conserved tyrosine kinase regions (Finnerty et al., supra; PCT/US93/00586, supra; Aprelikova et al., Cancer Res., 52: 746-748 [1992]) or by low-stringency hybridization with a Flk2 probe. Galland et al., Genomics, 13: 475-478 (1992). Alternative splicing of the Flt4 mRNA produces two variants of the protein differing by 65 amino acids at the C-terminus. Pajusola et al., Oncogene, 8: 2931-2937 (1993). These variants migrate as bands of 170-190 kDa that are partially cleaved proteolytically in the extracellular domain to produce a form of about 125 kDa. Pajusola et al., Oncogene, 8, supra; Pajusola et al., Oncogene, 9: 3545-3555 (1994). Expression of the longer spliced form of Flt4 as a chimera with the extracellular domain of the CSF-1 receptor shows that the Flt4 intracellular domain can signal a ligand-dependent growth response in rodent fibroblasts. Pajusola et al., Oncogene, 9, supra; Borg et al., Oncogene, 10: 973-984 (1995). Flt4 has been localized to human chromosome 5q34-q35 (Aprelikova et al., supra; Galland et al., Genomics, supra); Flt1 and Flk1 are located at 13q12 (Imbert et al., Cytogenet. Cell Genet., 67: 175-177 [1994]) and 4q12. Sait et al., Cytogenet. Cell Genet., 70: 145-146 (1995); Spritz et al., Genomics, 22: 431-436 (1994).
VEGF is a homodimeric, cysteine-rich protein that can occur in at least four forms due to alternative splicing of its mRNA. Ferrara et al., supra. While VEGF is a high-affinity ligand for Flt1 and Flk1, it does not bind or activate Flt4. Pajusola et al., Oncogene, 9, supra. The only other closely related member of the VEGF family is placental growth factor (PlGF), which has 47% amino acid identity with VEGF. Maglione et al., Proc. Natl. Acad. Sci. USA, 88: 9267-9271 (1991). PlGF also occurs in two alternatively spliced forms which differ in the presence or absence of a basic heparin binding domain of 21 amino acids. Maglione et al., Oncogene, 8: 925-931 (1993); Hauser and Weich, Growth Factors, 9: 259-268 (1993). PlGF binds to Flt1 but not to Flk1 (Park et al., J. Biol. Chem., 269: 25646-25654 [1994]); it is believed that its binding to Flt4 has not been determined. PlGF fails to duplicate the capillary endothelial cell mitogenesis or vascular permeability activities of VEGF, suggesting that these activities are mediated by the Flk1 receptor. Park et al., supra.
Molecules that modulate the Flk1 receptor or neutralize activation of a VEGF receptor are disclosed in the patent literature. For example, WO 95/21613 published Aug. 17, 1995 discloses compounds that modulate KDR/Flk1 receptor signal transduction so as to regulate and/or modulate vasculogenesis and angiogenesis and disclose using Flk1 to evaluate and screen for drugs and analogs of VEGF involved in Flk1 modulation by either agonist or antagonist activities; WO 95/21865 published Aug. 17, 1995 discloses molecules immunointeractive with animal neuroepithelial kinase (NYK)/Flk1, which molecules can be used to provide agents for treatment, prophylaxis, and diagnosis of an angiogenic-dependent phenotype; and WO 95/21868 published Aug. 17, 1995 discloses monoclonal antibodies that specifically bind to an extracellular domain of a VEGF receptor and neutralize activation of the receptor.
cDNA clones have now been identified that encode a novel protein, designated VRP, which binds to and stimulates the phosphorylation of the receptor tyrosine kinase Flt4. VRP is related in amino acid sequence to VEGF, but does not interact appreciably with the VEGF receptors, Flt1 and Flk1.
In one aspect, the invention provides isolated biologically active human VRP containing at least 265 amino acids. In another aspect, the invention supplies isolated biologically active human VEGF-related protein (VRP) comprising an amino acid sequence comprising at least residues +1 through 29, inclusive, of FIG. 1. In further aspect, the invention supplies isolated biologically active human VRP comprising an amino acid sequence shown as residues xe2x88x9220 through 399, inclusive, or residues 1 through 399, inclusive, of FIG. 1.
The invention also pertains to chimeras comprising the VRP fused to another polypeptide. For example, the invention provides a chimeric polypeptide comprising the VRP fused to a tag polypeptide sequence. An example of such a chimera is epitope-tagged VRP.
In another aspect, the invention provides a composition comprising biologically active VRP and a pharmaceutically acceptable carrier. In a more specific embodiment, the invention provides a pharmaceutical composition useful for promotion of vascular or lymph endothelial cell growth comprising a therapeutically effective amount of the VRP in a pharmaceutically acceptable carrier. In another aspect, this composition further comprises another cell growth factor such as VEGF and/or PlGF.
In a further aspect, the invention provides a method of treating vascular tissue and promoting angiogenesis in a mammal comprising administering to the mammal an effective amount of the composition comprising VRP. In another embodiment, the invention provides a method for treating trauma affecting the vascular endothelium comprising administering to a mammal suffering from said trauma an effective amount of the composition containing the VRP. The trauma is, for example, diabetic ulcers or a wound of the blood vessels or heart. In another embodiment, the invention provides a method for treating a dysfunctional state characterized by lack of activation or lack of inhibition of a receptor for VRP in a mammal comprising administering to the mammal an effective amount of the composition containing the VRP.
The invention also provides a method which involves contacting the Flt4 receptor with the VRP to cause phosphorylation of the kinase domain thereof. For example, the invention provides a method for stimulating the phosphorylation of a tyrosine kinase domain of a Flt4 receptor comprising contacting an extracellular domain of the Flt4 receptor with the VRP.
The invention also provides a monoclonal antibody which binds to the VRP and preferably also neutralizes a biological activity of the protein, one biological activity being characterized as promoting neovascularization or vascular permeability or vascular endothelial cell growth in a mammal. Alternatively or conjunctively, the invention provides a monoclonal antibody which binds to the N-terminal portion from residues xe2x88x9220 through 137, inclusive, or from residues +1 through 137, inclusive, of the amino acid sequence shown in FIG. 1. The antibody can be used, for example, to detect the presence of the VRP in a biological sample suspected of having the protein, or to treat patients. The invention contemplates a pharmaceutical composition comprising such antibody and a pharmaceutically acceptable carrier, as well as a method of treating diseases or disorders characterized by undesirable excessive neovascularization or vascular permeability in a mammal comprising administering to said mammal an effective amount of one of the antibodies described above. Further included by the invention is a method for treating a dysfunctional state characterized by excessive activation or inhibition of a receptor for VRP in a mammal comprising administering to the mammal an effective amount of one of the antibodies described above.
In addition, the invention contemplates a peptide consisting of an amino acid sequence shown as residues xe2x88x9220 through xe2x88x921, inclusive, of FIG. 1.
In a further embodiment, the invention provides an isolated nucleic acid molecule encoding VRP or a VRP chimera. In one aspect, the nucleic acid molecule is RNA or DNA that encodes a biologically active VRP or is complementary to nucleic acid sequence encoding such VRP, and remains stably bound to it under stringent conditions. The nucleic acid molecule optionally includes the regions of the nucleic acid sequences of FIG. 1 which encode signal sequences. In one embodiment, the nucleic acid sequence is selected from:
(a) the coding region of the nucleic acid sequence of FIG. 1 that codes for the preprotein from residue xe2x88x9220 to residue 399 or that codes for the mature protein from residue 1 to residue 399 (i.e., nucleotides 372 through 1628, inclusive, or nucleotides 432 through 1628, inclusive, of the nucleic acid sequence shown in FIG. 1 as SEQ ID NO: 1); or
(b) a sequence corresponding to the sequence of (a) within the scope of degeneracy of the genetic code.
In another aspect, the nucleic acid molecule can be provided in a replicable vector comprising the nucleic acid molecule operably linked to control sequences recognized by a host cell transfected or transformed with the vector. The invention further provides a host cell comprising the vector or the nucleic acid molecule. A method of producing VRP is also provided which comprises culturing a host cell comprising the nucleic acid molecule and recovering the protein from the host cell culture.